Project Summary/Abstract The dopamine system has a diverse repertoire of functions in the CNS including in voluntary motor control and reward. Mixtures of inhibitory and excitatory inputs overlaid on pacemaker firing of midbrain dopamine neurons controls circuit function. One such controlling factor is lateral inhibition by somatodendritic release of dopamine onto neighboring SNc and VTA dopamine neurons. Dopamine activates inhibitory D2-autoreceptors which couple to GIRK channels. The level of D2 sensitization controls the magnitude of inhibition generated by dopamine release. Little is known about the mechanism of D2 receptor desensitization except that calcium entry has been demonstrated to accelerate desensitization. This is highlighted by recent work which implicates non-canonical mechanisms of G-protein-coupled-receptor (GPCR) desensitization. This proposal seeks to further characterize D2 receptor regulation with two specific aims using whole-cell voltage-clamp recordings of SNc dopamine neurons in acute slice. First, what is the time course and calcium sensitivity of recovery from desensitization? Preliminary findings indicate recovery from desensitization is a rapid process, with signaling returning to baseline in about five minutes. This contrasts with experiments done with similar receptors, such as the -opioid receptor, which takes 30 or more minutes to recover. However, additional data are needed to uncover any calcium-dependence of desensitization as well as determine the relationship, if any, between degree of desensitization and time course of recovery. Aim two focuses on the receptor occupancy requirements for D2 receptor signaling; does an individual receptor require a bound agonist for downstream signaling to desensitize? This aim will be pursued by three sub-questions and, once again, how calcium might modify outcomes will be tested throughout these experiments. Does a low concentration of dopamine pre-desensitize the response to a high concentration of dopamine? In preliminary experiments, low concentrations of dopamine (~500 nM) desensitize the response to a high concentration test pulse. Does activation of other GPCRs desensitize D2 receptors? Though the results must be extended to other GPCRs, initial experiments have indicated pronounced heterologous desensitization with by activation of the inhibitory GPCR GABAB receptors. And finally, can desensitization spread from an initial location? The study of D2 signaling in specific compartments has remained inaccessible due to technological limitations. The recent production of CyHQ-O-DA, a photoactivatable caged dopamine with the ability to be photolyzed by 2-photon emission, allows for the spatially defined release of dopamine. This will allow for the induction of desensitization by repeated uncaging on a focal point and the probing to what degree the desensitization is localized by uncaging at various locations along dendrites and soma.